Abstract
Pancortin (PCT), a protein highly expressed in the cortex during neurodevelopment, comprises four isoforms (PCT1–4) characterized by a central B-region and N-termini (A1/A2) and C-termini (C1/C2). While PCT2, enriched in adult mouse cortex, mediates ischemic neuronal death via interactions with WAVE1 and Bcl-xL, perinatal isoforms (A2-PCTs, namely PCT3 and PCT4) remain poorly defined. Given the vulnerability of the developing brain to hypoxia-induced neurological deficits, here we asked whether A2-PCTs contribute to ischemic damage in the developing cortex. In primary cortical neurons, knockdown of PCTs mitigated cell death induced by oxygen-glucose deprivation. Consistently, using A2-PCTs knockout mice subjected to middle cerebral artery occlusion, we observed significantly reduced cortical damage in younger animals, implicating A2-PCTs as pro-death factors in hypoxic conditions. Mechanistically, A2-PCTs formed a tripartite complex with Bcl-xL and WAVE1. Overexpression of A2-PCTs in HEK293 cells resulted in elevated cell mortality, and as revealed by SPLICS (a split-GFP-based contact site sensor)-based imaging of mitochondria-ER contact sites (MERCs), increased its localization to MERCs. Furthermore, A2-PCTs interacted with GRP75, a MERCs-tethering protein, in a Bcl-xL/WAVE1-dependent manner. A2-PCTs/Bcl-xL/WAVE1 complex increased IP3R-mediated calcium transfer from the ER to mitochondria, leading to cytosolic and mitochondrial calcium overload, which was attenuated by IP3R inhibition. In Neuro2a cells subjected to oxygen-glucose deprivation, PCTs knockdown similarly suppressed pathological calcium flux. Our study identifies A2-PCTs as key regulators of MERCs-mediated calcium dysregulation in neonatal stroke, pointing to their potential as therapeutic targets for mitigating ischemic brain injury in the developing brain.
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The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
We extend our sincere gratitude to all members of the research team for their invaluable contributions and collaborative efforts throughout this study, especially those who worked for this project at the beginning, such as Takashi Nagano, Mitsuyo Maeda, and Hiroshi Takagi. We thank the Genome Information Research Center, Research Institute for Microbial Diseases (RIMD), The University of Osaka, for their assistance in generating the knockout mice. We would like to express our gratitude to the Center for Medical Research and Education (CentMERe), The University of Osaka, for providing access to and assistance with microscopy services. We also thank The Center of Medical Innovation and Translational Research (CoMIT) for performing the sequencing and for their support with the OGD experiments. Their contributions were essential to this work. We thank Jia-Xuan Li for assistance with Western blotting, Ming-Yue Ma for establishing and maintaining neuronal cultures, Guo-Qiang Yang and Zhen-Ying Zhang for quantitative analysis of cell death, and Nikita Diubachev, Lan Yang, and Ghita Bennis for quantification of cellular staining signals.
Funding
This work was supported in part by Naito Foundation, Senri-Life Science Foundation, and the Ministry of Education, Science, Sports and Culture of Japan (to M.S., grand IDs: 20H03414, 15K15015, 25293043, 19390048, 11878150, 07780686).
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Q.Y.: methodology, investigation (experimental design and execution), formal analysis, resources, data curation, original manuscript; C.W.: methodology (molecular engineering), resources (plasmid constructs: pCAGGS-PCT, pCAGGS-WAVE1, pCAGGS-Bcl-xL), formal analysis (MCAO statistical datasets); M.X., T.M.: investigation (MCAO surgery); H.Y.: methodology (murine knockout models), manuscript revision, project administration; M.Y.: manuscript revision, supervision, discussion, validation, methodology (technical guidance); C.T., Y.O.: manuscript revision, methodology (technical guidance), supervision, discussion; T.M., K.K.: project administration; M.S: project administration, manuscript revision, supervision, discussion, initial conceptualization and experimental design.
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Yang, Q., Wang, CC., Matsuyama, T. et al. A2-pancortins interact with Bcl-xL and WAVE1 to promote mitochondria-ER contact sites (MERCs) and exacerbate mitochondrial calcium elevation to mediate cell death in stroke. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38928-3
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DOI: https://doi.org/10.1038/s41598-026-38928-3
